Gene expression is regulated, in part, by the cellular processes involved in transcription. During transcription, a single-stranded RNA complementary to the DNA sequence to be transcribed is formed by the action of RNA polymerases. Initiation of transcription in eukaryotic cells is regulated by complex interactions between cis-acting DNA motifs, located within the gene to be transcribed, and trans-acting protein factors. Among the cis-acting regulatory regions are sequences of DNA, termed promoters, to which RNA polymerase is first bound, either directly or indirectly. As used herein, the term “promoter” refers to the 5′ untranslated region of a gene that is associated with transcription and which generally includes a transcription start site. Other cis-acting DNA motifs, such as enhancers, may be situated further up- and/or down-stream from the initiation site.
Both promoters and enhancers are generally composed of several discrete, often redundant elements, each of which may be recognized by one or more trans-acting regulatory proteins, known as transcription factors. Promoters generally comprise both proximal and more distant elements. For example, the so-called TATA box, which is important for the binding of regulatory proteins, is generally found about 25 basepairs upstream from the initiation site. The so-called CAAT box is generally found about 75 basepairs upstream of the initiation site. Promoters generally contain between about 100 and 1000 nucleotides, although longer promoter sequences are possible.
To date, although numerous promoters have been isolated from various plants, only a few of these are usefully employed for expression of a transgene in a plant. Currently CaMV (cauliflower mosaic virus) 35S promoter and its derivatives have been most widely used. This promoter is constitutive, i.e. continuously active in all plant tissues. However, the CaMV 35S promoter exhibits lower activity in monocot plants, such as rice and maize, than in dicot plants, and does not exhibit any activity in certain cells such as pollen. Many other promoters that have originated from dicot plants have also been used for transgene expression in monocot plants, but exhibit lower activity than promoters originating from monocot plants.
Intron sequences inside monocot promoters have been shown to enhance promoter activity. These include the first intron of rice actin (McEloy et al., Mol. Gen. Genet. 231:150-160, 1991), intron 1 of the maize ubiquitin gene (Christensen and Quail, Transgenic Res. 5:213-218, 1996), and the maize sucrose synthase gene (Clancy and Hannah, Plant Physiol. 130:918-929, 2002). Using the actin intron next to the 35S promoter increased expression 10-fold in rice, compared to 35S promoter alone (McElroy et al., Mol. Gen. Genet. 231:150-160, 1991). Studies have shown that the introns used must be within the transcribed portion of the gene and preferably within the 5′ untranslated leader sequence (Bourdon et al., EMBO Rep. 2:394-398, 2001; Callis et al., Genes Dev. 1:1183-1200, 1987; Mascarenhas et al., Plant Mol. Biol. 15:913-920, 1990). It has also been shown that the intron plays a role in tissue specificity in some cases (Deyholos and Sieburth, Plant Cell 12:1799-1810, 2000).
In addition to introns, untranslated leader sequences (5′UTLs) have also been shown to enhance expression. It appears that 5′UTLs from dicots work better in dicot hosts and those from monocots work better in monocots (Koziel et al., Plant Mol. Biol. 32:393-405, 1996).
Constitutive promoters have been isolated from monocots, characterized, and used to drive transgene expression, for example the rice actin1 promoter and the maize ubiquitin 1 promoter. However, even within monocots, using a promoter in a heterologous system may give unexpected expression patterns. For example, the rbcS promoter from rice has a different pattern of expression than the endogenous maize rbcS when transformed into a maize plant (Nomura et al., Plant Mol. Biol. 44:99-106, 2000). Therefore, there is a need for the development of promoter systems from monocots and, in particular, important target species such as forage grasses.
Constitutive promoters for use in monocots, especially the forage grasses, are not abundant. Examples of these may be promoters from the genes of actin, tubulin or ubiquitin. Actin is a fundamental cytoskeletal component that is expressed in nearly every plant cell. The alpha- and beta-tubulin monomers associate to form tubulin dimers that are the basic units of microtubules, found in most cells. Ubiquitin is one of the most highly conserved proteins in nature. It has been linked to many cellular processes such as protein degradation, chromatin structure and DNA repair, and is highly abundant in nearly every plant cell (Kawalleck et al., Plant Mol. Biol. 21:673-684, 1993).
In some cases, constitutive over-expression of a transgene may interfere with the normal processes in a plant. The development of tissue-specific promoters, designed specifically to drive a particular gene of interest should help to alleviate these problems. For example, to manipulate the plant secondary cell wall, vascular specific promoters may be preferred, and to manipulate flowering habit, floral specific promoters may be preferred.
A number of genes in the pathway for lignin biosynthesis from Lolium perenne and Festuca arundinacea are described in International Patent Publications WO03/040306 and WO03/93464. These include Phenylalanine Ammonia Lyase (PAL), the first enzyme of the general phenylpropanoid pathway. Isoforms of this gene from Arabidopsis have been shown to be stem and vascular specific in expression (Ohl et al., Plant Cell 2:837-848, 1990; Leyva et al., Plant Cell 4:263-271, 1992). Several isoforms of 4-Coumarate:CoA ligase (4CL) have been isolated. 4CL is an enzyme that catalyzes the formation of CoA esters from p-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid and sinapic acid. A number of caffeic acid O-methyltransferase (COMT) grass genes have also been identified. COMT genes, such as those from Arabidopsis and the monocot alfalfa, are expressed in lignifying tissues (Goujon et al., Plant Mol. Biol. 51:973-989, 2003; Inoue et al., Plant Physiol. 117:761-770, 1998). Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis, and the grass CAD gene has also been identified. The promoters of these genes will be of use in manipulating cell wall modification and digestibility.
A number of genes involved in flowering development from Lolium perenne and Festuca arundinacea are described in International Patent Publication WO04/022755. The control of flowering has been extensively studied in model species, in particular Arabidopsis thaliana, and a large number of genes and transcription factors involved in floral development have been identified; for a review see Putterill et al., BioEssays 26:363-373, 2004, and Simpson & Dean, Science 296:285-289, 2002. In particular, the MADs box family of transcription factors play a role in the transition of vegetative to floral growth and show differential expression through floral development (Petersen et al., J. Plant Physiol. 161:439-447, 2004. In the manipulation of floral development, it is a prerequisite that floral specific promoters will be required to drive transgene expression. Therefore, the isolation and development of floral specific promoters from monocots is necessary.
A number of genes involved in anthocyanin and condensed tannin biosynthesis from Lolium perenne and Festuca arundinacea are described in International Patent Publications WO03/040306 and WO03/93464. Many of the genes involved in anthocyanin biosynthesis show specific cell type and developmental patterns of expression. The promoters of these genes will be of use in transgenic expression of genes, particularly to manipulate anthocyanin and tannin biosynthesis. Dihydroflavonol-4-Reductase (DFR) catalyzes the reduction of dihydroflavonols to leucoanthocyanidins, the precursors of anthocyanins and condensed tannins. DFR is a later key enzyme that may control the flux into the pathways of anthocyanin and condensed tannin synthesis. Another key enzyme that may control flux into these pathways is chalcone synthase (CHS), which catalyzes the condensation of malonyl-CoA and coumaroyl-CoA into chalcone intermediates. In many species, several gene family members exist for each enzyme. These different family members are differentially expressed and reflect the types of tissue in which different species accumulate anthocyanins, such as fruit or petals (Jaakola et al., Plant Physiol. 130:729-739, 2002; Rosati et al., Plant Mol. Biol. 35:303-311, 1997). In particular, grasses accumulate higher levels of anthocyanins in the stem.
A number of antifreeze protein genes from Lolium perenne and Festuca arundinacea are described in International Patent Publication WO04/022700. Overwintering plants produce antifreeze proteins (AFPs) having the ability to adsorb onto the surface of ice crystals and modify their growth. AFPs may play a role in protecting the plant tissues from mechanical stress caused by ice formation (Atici and Nalbantoglu, Phytochem. 64:1187-1196, 2003). The expression of AFPs is induced by cold temperature, in specific plant tissues, and a system utilizing these specific promoters will be very powerful.
A number of fructosyltransferase genes from Lolium perenne and Festuca arundinacea are described in International Patent Publication WO 03/040306. Fructosyltransferases catalyze the synthesis of fructans, polymers of fructose found in a range of plant families including the Poaceae. Fructans are found in specific organs dependent on the plant species. In the grasses they are found in the stems and leaf base where expression of specific fructosyltransferases occurs (Luscher et al., Plant Physiol. 124:1217-1227, 2000). The promoters of these genes will be useful to drive specific expression of transgenes.
Plants produce a number of Class III plant peroxidase (POX) enzymes, and each isoenzyme has diverse expression profiles, suggesting their involvement in various physiological processes (for a review see Hiraga et al., Plant Cell Physiol. 42:462-468, 2001). POXs have been suggested to play a role in lignification, suberization, auxin catabolism, wound healing and defense against pathogen infection. The unique expression profile of these genes, captured by isolation of their promoters will provide a valuable tool for expression of transgenes.